Difference between revisions of "Team:Virginia/Results"

 
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          <div class="sectionTitle" id="Section 1">Section 1</div>
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                The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) with encapsulated dsDNA scaffolds <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div> that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
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              The invention consists of a protein shell comprising one or more proteins, one or more nucleic acid scaffolds of which there can be multiple copies, anabolic and/or catabolic enzymes specific to the desired biosynthesis pathway each containing a nucleic acid binding domain, recognition sequences for the utilized nucleic acid binding domains, nucleic acid spacers, and a linkage between the nucleic acid scaffolds and the protein shell. The protein shell (10) can take the form of any closed or open surface that comprises one or more repeating protein units (12). Examples of valid shells include bacterial microcompartments such as the Pdu, Eut, and carboxysome microcompartments, as well as modified,  but not necessarily closed, surfaces composed of mutated versions of these microcompartment shell proteins. The nucleic acid scaffolds (18) comprise multiple recognition sequences (22) and spacers (32) and can be made from any form of nucleic acid, including: deoxyribonucleic acid, ribonucleic acid, and synthetic nucleic acids such as xeno nucleic acids and peptide nucleic acids among others. These scaffolds are attached to the protein shell. The pathway enzymes are biological proteins whose exact sequences are dependent on the given use case of the invention, but which all contain a nucleic acid binding domain either internal to their structure, or at their N or C terminus.
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              <b>Fig 1.</b> Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.
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              Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used,  if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).<br/><br/><br/>
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Inspiration">Inspiration</a>
 
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Design">Design</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Results">Results</a>
 
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Entrepreneurship">Entrepreneurship</a>
 
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              <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/Gallery">Gallery</a>
 
 
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             <a class="mainitem" href="#about">RESOURCES</a>
 
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               <a class="hvr-sweep-to-right" href="https://2020.igem.org/Team:Virginia/nucleic_acids">Nucleic Acids</a>
 
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             <div>Results</div>
 
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           <div class="sectionTitle" id="Section 1">Section 1</div>
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           <div class="sectionTitle" id="Section 1">Summary of Results</div>
 
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                 The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) with encapsulated dsDNA scaffolds <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div> that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
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                 With only 5 weeks of lab, there was a rush to verify sequences with restriction enzyme digests, build any of the preliminary parts, and visualize <i>E. coli</i> with BMCs in them. Unfortunately, the time limitation prevented us from succesfully completing most of the steps we planned for in the last 10 months, but shown below are results from one series of procedures we performed to produce BMCs in <i>E. coli</i> BL21(DE3). With the generous help of Dr. Criswell at UVA, <b>we were able to successfully culture the cells, fixate them, and take pictures of 6 different samples using a Transmission Electron Microscope (TEM).</b>
 
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           <div class="sectionTitle" id="Section 3">TEM Set-Up</div>
 
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               The invention consists of a protein shell comprising one or more proteins, one or more nucleic acid scaffolds of which there can be multiple copies, anabolic and/or catabolic enzymes specific to the desired biosynthesis pathway each containing a nucleic acid binding domain, recognition sequences for the utilized nucleic acid binding domains, nucleic acid spacers, and a linkage between the nucleic acid scaffolds and the protein shell. The protein shell (10) can take the form of any closed or open surface that comprises one or more repeating protein units (12). Examples of valid shells include bacterial microcompartments such as the Pdu, Eut, and carboxysome microcompartments, as well as modified,  but not necessarily closed, surfaces composed of mutated versions of these microcompartment shell proteins. The nucleic acid scaffolds (18) comprise multiple recognition sequences (22) and spacers (32) and can be made from any form of nucleic acid, including: deoxyribonucleic acid, ribonucleic acid, and synthetic nucleic acids such as xeno nucleic acids and peptide nucleic acids among others. These scaffolds are attached to the protein shell. The pathway enzymes are biological proteins whose exact sequences are dependent on the given use case of the invention, but which all contain a nucleic acid binding domain either internal to their structure, or at their N or C terminus.  
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               The series of TEM Setup procedures are described in the Notebook page for 10/20 and 10/21. We transfected the   BL21(DE3) cells with with pdu-ABJKNU and pdu-ABJKNUT, and then prepared 5 plates as follows:
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              <ol>
 +
                <li>BL21(DE3) control </li>
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                <li>pdu-ABJKNU </li>
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                <li>pdu-ABJKNU, diluted cell concentration </li>
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                <li>pdu-ABJKNUT</li>
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                <li>pdu-ABJKNUT, diluted cell concentration</li>
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              </ol>
 +
              Plates 1, 2, and 5 are pictured below and were the cultured cells we used for preparing TEM samples. As seen in Figure 1, the higher concentration of pdu-ABJKNU cells grew at a similar rate to the control cells and the diluted pduABJKNUT.
 
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               <b>Fig 1.</b> Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.
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               <i><b>Fig 1: Plates for TEM</b> [A] shows the Untransformed BL21(DE3) control strain. [B] shows the Transformed pdu-ABKJNU cells on plate 2. [C] shows the Transformed pdu-ABJKNUT cells on plate 5.</i>
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          <div class="sectionTitle" id="Section 2">TEM Pictures</div>
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               Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used,  if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).<br/><br/><br/>
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               The following 6 cultures were fixated for TEM use as follows:
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              <ol>
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                <li>BL21(DE3)</li>
 +
                <li>BL21(DE3) + IPTG</li>
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                <li>pdu-ABJKNU</li>
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                <li>pdu-ABJKNU + IPTG</li>
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                <li>pdu-ABJKNUT</li>
 +
                <li>pdu-ABJKNUT + IPTG</li>
 +
              </ol>
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            A sample picture has been selected from each of the samples and is shown in Figure 2. We were not able to determine any significant difference between the samples, suggesting that there was a problem with the production of BMCs. The most likely reason for this is a deviation from the protocol we intended to follow due to a time limitation. Instead of allowing the samples to sit at room temperature overnight, we only allowed them to sit for a few hours, which led us to fix the cells before enough time for BMC formation had passed.  
 
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              <b><i>Fig 2: E. coli under TEM</i></b> Top left is a sample from Culture 1 (Untransformed), top center is a sample from Culture 2 (Untransformed + IPTG), top right is a sample from Culture 3 (Transformed with pduABJKNU), bottom left is a sample from Culture 4 (Transformed with pduABJKNU + IPTG), bottom center is a sample from Culture 5 (Transformed with pduABJKNUT), and bottom right is a sample from Culture 6 (Transformed with pduABJKNUT + IPTG). Open each image in a new tab to view it at full scale. The whole collection of images is available <a href="https://static.igem.org/mediawiki/2020/e/e1/T--Virginia--media--TEM_Results.pdf">here</a>.
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           <div class="menulogo">MANIFOLD</div>
 
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Latest revision as of 03:32, 28 October 2020

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Results
Summary of Results
With only 5 weeks of lab, there was a rush to verify sequences with restriction enzyme digests, build any of the preliminary parts, and visualize E. coli with BMCs in them. Unfortunately, the time limitation prevented us from succesfully completing most of the steps we planned for in the last 10 months, but shown below are results from one series of procedures we performed to produce BMCs in E. coli BL21(DE3). With the generous help of Dr. Criswell at UVA, we were able to successfully culture the cells, fixate them, and take pictures of 6 different samples using a Transmission Electron Microscope (TEM).
TEM Set-Up
The series of TEM Setup procedures are described in the Notebook page for 10/20 and 10/21. We transfected the BL21(DE3) cells with with pdu-ABJKNU and pdu-ABJKNUT, and then prepared 5 plates as follows:
  1. BL21(DE3) control
  2. pdu-ABJKNU
  3. pdu-ABJKNU, diluted cell concentration
  4. pdu-ABJKNUT
  5. pdu-ABJKNUT, diluted cell concentration
Plates 1, 2, and 5 are pictured below and were the cultured cells we used for preparing TEM samples. As seen in Figure 1, the higher concentration of pdu-ABJKNU cells grew at a similar rate to the control cells and the diluted pduABJKNUT.
Fig 1: Plates for TEM [A] shows the Untransformed BL21(DE3) control strain. [B] shows the Transformed pdu-ABKJNU cells on plate 2. [C] shows the Transformed pdu-ABJKNUT cells on plate 5.

TEM Pictures
The following 6 cultures were fixated for TEM use as follows:
  1. BL21(DE3)
  2. BL21(DE3) + IPTG
  3. pdu-ABJKNU
  4. pdu-ABJKNU + IPTG
  5. pdu-ABJKNUT
  6. pdu-ABJKNUT + IPTG
A sample picture has been selected from each of the samples and is shown in Figure 2. We were not able to determine any significant difference between the samples, suggesting that there was a problem with the production of BMCs. The most likely reason for this is a deviation from the protocol we intended to follow due to a time limitation. Instead of allowing the samples to sit at room temperature overnight, we only allowed them to sit for a few hours, which led us to fix the cells before enough time for BMC formation had passed.

Fig 2: E. coli under TEM Top left is a sample from Culture 1 (Untransformed), top center is a sample from Culture 2 (Untransformed + IPTG), top right is a sample from Culture 3 (Transformed with pduABJKNU), bottom left is a sample from Culture 4 (Transformed with pduABJKNU + IPTG), bottom center is a sample from Culture 5 (Transformed with pduABJKNUT), and bottom right is a sample from Culture 6 (Transformed with pduABJKNUT + IPTG). Open each image in a new tab to view it at full scale. The whole collection of images is available here.


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